The present invention relates generally to compositions and methods involving aminophospholipids, and more particularly to pH-dependent caged aminophospholipids, liposomes formed with the caged aminophospholipids, and pharmaceutical preparations containing such liposomes.
As further background, all biological membranes display an asymmetric transmembrane orientation of the four major classes of phospholipids. Generally, the amine-containing phospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE) are enriched in the cytofacial monolayer, while the choline-containing phospholipids, phosphatidylcholine (PC) and spingomyelin (SM) are found on the opposite face of the membrane (Bretscher, 1973; Verkleij et al., 1973; Rothman & Lenard, 1977). This asymmetry is maintained by slow passive transmembrane flip-flop (Kornberg & McConnell, 1971; Van Meer & Op den Kamp, 1982; Homan & Pownall, 1988; Middlekoop et al., 1986), binding of acidic lipids to inner monolayer proteins (Haest et al., 1978; Franck et al., 1985) and by the selective, ATP-dependent, inward transport of aminophospholipids (Devaux, 1991; Schroit & Zwaal, 1991).
The aminophospholipid transporter, or "flippase," requires Mg2+-ATP and is inhibited by vanadate, calcium, and sulfhydryl reagents (Seigneuret & Devaux, 1984; Daleke & Huestis, 1985; Connor & Schroit, 1987; Bitbol et al., 1987). The enzyme displays an absolute specificity for lipid headgroup structure. The flippase transports amine-containing phospholipids exclusively and has an affinity for PS approximately ten-fold greater than for PE (Zachowski et al., 1986). Alkylating the amine function, esterifying the carboxyl group, or increasing the distance between the phosphate and carboxyl/amine groups results in reduced flippase activity (Morrot et al., 1989; E. Nemergut, M. Zimmerman and D. Daleke, unpublished observations). Transport is dependent on the configuration of the glycerol backbone. Removing the sn-2 fatty acyl group (Daleke & Huestis, 1985), inverting the stereochemistry in the sn-2 position (Martini & Pagano, 1987), or replacing the diacylglycerol moiety with ceramide (Morrot et al., 1989) reduces considerably the transport of aminophospholipids. In contrast, transport activity is relatively insensitive to acyl chain length (Daleke & Huestis, 1985), although hydrophilic fluorescent fatty acyl substituents result in reduced transport rates (Colleau et al., 1991). The relative insensitivity of the flippase to acyl chain length and composition has allowed the design of PS substrates labeled with a variety of acyl chain reporter groups. In addition to short (Daleke & Huestis, 1985) and long (Tilley et al., 1986) chain radiolabeled fatty acids, spin label (Seigneuret & Devaux, 1984) and fluorescent (Connor & Schroit, 1987) fatty acids have been used to measure flippase activity. The strategy behind the design of these labeled lipids is that they are relatively hydrophilic and will rapidly and spontaneously exchange from donor vesicles to cell membranes. The transmembrane distribution is subsequently assessed by extracting outer monolayer lipid into albumin or exogenous acceptor vesicles. The amount of spin, fluorescent, or radiolabeled lipid remaining in the cell is considered the amount of lipid transported to the inner monolayer.
The measurement of transport kinetics required that the rate of lipid incorporation be faster than the rate of transport. These measurements have been confounded by concurrent incorporation and transport of lipid probes. One solution is prior inhibition of the flippase by using a reversible inhibitor such as vanadate, a sulfhydryl reagent, or low temperatures. This obviates the need for rapid lipid incorporation, however these methods are complicated by relatively slow reversal of inhibition (sulfhydryl reagents) or slow lipid incorporation (low temperatures).
In another facet, the use of liposomes as drug delivery systems has been explored for some time. A primary problem in liposomal delivery has been the inability to effectively release liposomal contents into the cell cytoplasm: most liposomes are taken up by cells into endosomes, low pH membrane compartments within the cell. To overcome these problems, the applicant has designed and synthesized reversibly N-modified or "caged" aminophospholipids. These lipids are N-acylated with structural analogs of maleic anhydride, which can be released by exposure to low pH solutions. The lipids can be incorporated into the liposomes to create pH-sensitive liposomes which can be used to effectively deliver active agents such as pharmaceuticals to cellular cytoplasm; and can also be used in labeling cells and the study of enzymatic activity such as flippase activity.